ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis

Background & Aims: Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown. Methods: Hepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development. Results: Hepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH. Conclusions: Despite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset.


Supplementary Methods
In vivo treatments 2 mo male Atf4 F/F , Atf4 ∆hep , MUP-uPA/Atf4 F/F , and MUP-uPA/Atf4 ∆hep mice were injected with AAV8-mCherry or AAV8-xCT (8 × 10 10 genome copies/mice) via the tail vein.MUP-uPA/Atf4 F/F and MUP-uPA/Atf4 ∆hep mice were fed with HFD from 3 mo, until 10 mo when sacrificed for the tumor analysis.LFD-fed AAV8-injected MUP-uPA/Atf4 ∆hep mice were sacrificed at 3.5 mo to analyze liver injury.AAV8-injected Atf4 F/F and Atf4 ∆hep mice were used for the HFD-fed DENinduced HCC model described above until sacrificed at 10 mo for sample collection.For acute DEN-induced liver injury, 2-3 mo male Atf4 F/F and Atf4 ∆hep mice were i.p. injected with 100 mg/kg DEN.After 24 and 48 hrs, livers and serum were collected.For FER-1 administered MUP-uPA/Atf4 ∆hep mice, 6-wo mice were i.p. injected with vehicle control DMSO or 1 mg/kg FER-1 every other day for 2 weeks before livers and serum were analyzed.Only male mice were used and the number of mice per experiment and their age are indicated in the figure legends.

Sample collection and Histology
Mouse liver was removed, weighed, and photographed before separation into individual lobes.
Large lobes were fixed in 10% formalin for 24-48 hrs to prepare paraffin blocks or embedded in Tissue-Tek OCT compound (Sakura Finetek) for frozen block preparation.Frozen tissue sections were stained with Oil Red O (ORO) to detect lipids or dihydroethidium (DHE) for ROS.Formalinfixed paraffin-embedded (FFPE) tissues were used for hematoxylin and eosin (H&E), Sirius Red staining, and IHC staining.Positively stained areas were quantified in 3-6 random fields (× 100, × 200, or × 400) on each slide using Image J software.The remaining lobes were dissected and stored at −80° C until analyzed.5 μm thick sections were stained with H&E and processed for IHC as described before [1].Briefly, deparaffinized and dehydrated sections were blocked with 5% goat serum for 1 hr at room temperature, primary antibodies for 1 hr at room temperature, followed by incubation with biotinylated secondary antibodies (1:200) for 30 min and streptavidin-HRP (1:500) for 30 min.
Serum ALT levels were measured using Infinity ALT (GPT) reagent (Thermo scientific TR71121) according to the supplied protocol.Liver triglycerides (TG), serum TG, and serum cholesterol were measured with a Triglyceride Colorimetric Assay Kit (Cayman Chemical 10010303) and a Cholesterol Fluorometric Assay Kit (Cayman Chemical #10007640), respectively, according to the manufacturer's protocol.Liver cholesterol was measured with a Cholesterol/Cholesteryl Ester Assay Kit (Abcam ab65359) according to the manufacturer's protocol.Liver and hepatocytes GSH and GSSG measurements were performed using GSH/GSSG-Glo™ Assays (Promega V6611) according to the manufacturer's protocol.TUNEL staining was performed using an in-situ cell death detection kit (Roche 12156792910).Images were captured on an upright light/fluorescent microscope (Zeiss) equipped with an AxioCam camera.

In Situ Hybridization (ISH)
For ATF4 ISH, ATF4 probes and a detection kit from RNAscope Probe-Mm-Atf4 (ACD Bio 405101) were used and mouse liver sections were stained according to the manufacturer's protocol RNAscope® 2.5 HD Assay-BROWN.
Infection with adenovirus was performed 6 hrs after seeding on the collagen-coated plates at a moi of 10.After 24 hrs infection, cells were further treated as indicated before being collected for protein and RNA analysis.
For isolation of tumor cells from mice, tumors were dissected from livers and cut into pieces of about 1 mm 3 , followed by digestion with liberase and removal of red blood cells (RBC) with 1XRBC lysis buffer (eBioscience 00-4300-54) before centrifugation and culturing.
All cells were incubated at 37 °C in a humidified chamber with 5% CO2.
In some experiments, trypan blue dye (Solarbio C0040) exclusion counting was performed.Cell viability under basal conditions is the viability of vehicle control.

Protein extraction and immunoblots
Livers were homogenized in a Dounce homogenizer (Thomas Scientific, NJ) with 30 strokes in RIPA buffer containing protease and phosphatase inhibitor cocktails.Primary hepatocytes were lysed directly in RIPA buffer containing protease and phosphatase inhibitor cocktails.After centrifugation, supernatants were separated by SDS-PAGE.Nuclear extraction was performed by using NE-PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, 78833) and cytosolic and nuclear factions were separated according to the manufacturer's protocol.Protein concentrations were quantified by Bradford assay (Biorad 5000006) and analyzed by SDS-PAGE and IB.Details of primary and secondary antibodies are provided in Supplementary Table S1.

RNA isolation and Quantitative real-time PCR (Q-RT-PCR)
Total RNA from liver tissue or primary hepatocytes was extracted using RNeasy Plus Mini kit (Qiagen Cat 74134) and cDNA was synthesized using SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific, 11754050).Q-RT-PCR was performed using SYBR green (Biorad 1725275) based QPCR on a Biorad CFX96 machine.Relative mRNA expression was calculated from the comparative cycle threshold (CT) values relative to Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) mRNA.Data are presented as arbitrary units and were calculated by the 2ˆ(-delta CT) method.Primer sequences were provided by Integrated DNA technologies and are listed in Supplementary Table S2.

Transmission Electron Microscopy (TEM)
Primary hepatocytes were seeded at a density of 4X10 6 cells/dish in 10-cm tissue culture dishes pre-coated with type-1 collagen (Rat tail, Corning 354236).Overnight cultured cells were treated with vehicle (DMSO) or RSL3 (2 μM) for 12 hrs.Cells were fixed with 2.5% glutaraldehyde in 0.1 M Phosphate buffer (PB, 0.1 M H2PO4, 0.1 M HPO4 [pH 7.2]) for at least 2 hrs, and then treated with 1% OsO4 in 0.1 M PB at 4 °C for 1 hr.Enblock staining used 2% aqueous uranyl acetate for 2 hrs at 4°C in dark.After dehydration through ethanol and acetone series, cells were embedded in pure epoxy resin.70 nm of sections were cut on a Leica ultramicrotome (Leica 705902), stained with 0.4% lead citrate, and examined under a FEI Tecnai G2 Spirit BioTWIN electron microscope (FEI, Hillsboro, OR, USA).Pictures were taken on a Gatan Digital image capturing system digital camera at 8,200-11,500-fold magnification at the Electron Microscopy Core Facility, Shanghai University of Traditional Chinese Medicine.

Adenovirus preparation
Adenoviruses expressing GFP, ATF4, or xCT were generated using pAdTrack-CMV and the AdEasy system as described [1].Adenoviruses were harvested and purified by CsCl ultracentrifugation and their titter was determined as described [1].

Adeno-associated virus type 8 (AAV8) preparation
PCR-amplified mouse xCT sequences were cloned into a pAAV-mCherry vector, a generous gift from Kang Zhang (University of California, San Diego).AAV8 was prepared as described [3].
F/F and MUP-uPA/Atf4 ∆ mice treated with different doses of ferroptosis inducer RSL3.Cell viability was assessed by CCK8 assay in triplicate and the effects of each treatment were normalized to the viability of vehicle control (NONE).